ab196995 II (Fluorometric CETP Activity Assay Kit...Ensure plates are properly sealed or covered during incubation steps. Ensure complete removal of all solutions and buffers from

Version 3 Last Updated 29 January 2020

Instructions for Use

For the rapid, sensitive and accurate measurement of CETP activity in animal plasma and serum.

This product is for research use only and is not intended for diagnostic use.

ab196995CETP Activity Assay Kit II (Fluorometric)

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Table of Contents

INTRODUCTION1. BACKGROUND 22. ASSAY SUMMARY 3

GENERAL INFORMATION3. PRECAUTIONS 44. STORAGE AND STABILITY 45. MATERIALS SUPPLIED 56. MATERIALS REQUIRED, NOT SUPPLIED 57. LIMITATIONS 68. TECHNICAL HINTS 7

ASSAY PREPARATION9. REAGENT PREPARATION 810. STANDARD PREPARATION 911. SAMPLE PREPARATION 10

ASSAY PROCEDURE and DETECTION12. ASSAY PROCEDURE and DETECTION 11

DATA ANALYSIS13. CALCULATIONS 1314. TYPICAL DATA 15

RESOURCES15. QUICK ASSAY PROCEDURE 1816. TROUBLESHOOTING 1917. FAQ 2118. INTERFERENCES 2219. NOTES 23


INTRODUCTION

1. BACKGROUND

CETP Activity Assay Kit II (Fluorometric) (ab196995) uses a donor molecule containing a fluorescent self-quenched phospholipid that is transferred to an acceptor molecule in the presence of CETP. CETP-mediated transfer of the fluorescent phospholipid to the acceptor molecule results in an increase in fluorescence (Ex/Em = 480/511 nm). The fluorometric intensity is directly proportional to the amount of phospholipid transferred. The kit contains rabbit serum and torcetrapib (CETP inhibitor) as positive and negative control, respectively, for assay validation.This assay can measure PLTP activity in plasma and serum but can also be used for testing activity of recombinant CETP protein.

Cholesteryl ester transfer protein (CETP) is a member of the lipid transfer/lipopolysaccharide binding protein gene family. CETP is plasma protein that transfers a cholesteryl ester from HDL to LDL or VLDL in exchange for a triglyceride. HDL plays an important role in lipid metabolism and cardiovascular health. HDL transports cholesterol to the liver for excretion or to steroidogenic tissues for steroid synthesis. HDL also plays an important role in the reverse cholesterol transport pathway, removing cholesterol from lipid-filled macrophages, protecting against atherosclerosis. Because of this function, CETP is viewed as a target to increase HDL, with CETP inhibition an active area of research and several CETP inhibitors at various stages of drug development.


INTRODUCTION

2. ASSAY SUMMARY

Standard curve preparation

Sample preparation

Add reaction mix and pre-incubate 37°C 30 min

Measure fluorescence (Ex/Em = 480/511 nm) in a

kinetic mode at 37°C for 1 -3 hours

*For kinetic mode detection, incubation time given in this summary is for guidance only.


GENERAL INFORMATION

3. PRECAUTIONSPlease read these instructions carefully prior to beginning the assay.All kit components have been formulated and quality control tested to function successfully as a kit. Modifications to the kit components or procedures may result in loss of performance.

4. STORAGE AND STABILITYStore kit at 4ºC in the dark immediately upon receipt, except from the CETP Positive Control which should be stored at -80°C. Kit has a storage time of 1 year from receipt, providing components have not been reconstituted.Refer to list of materials supplied for storage conditions of individual components. Observe the storage conditions for individual prepared components in section 5.Aliquot components in working volumes before storing at the recommended temperature. Reconstituted components are stable for 2 months.


GENERAL INFORMATION

5. MATERIALS SUPPLIED

Item AmountStorage

Condition(Before

Preparation)

StorageCondition

(After Preparation)

CETP Assay Buffer 20 mL 4°C 4°CCETP Donor Molecule (8 nmol/mL) 500 µL 4°C 4°CCETP Acceptor Molecule 500 µL 4°C 4°CCETP Positive Control (rabbit serum) 100 µL -80°C -80°C

CETP Inhibitor (1 mM) (Torcetrapib) 10 µL 4°C 4°C

6. MATERIALS REQUIRED, NOT SUPPLIEDThese materials are not included in the kit, but will be required to successfully perform this assay:

100% isopropanol

DMSO

Microcentrifuge

Pipettes and pipette tips

Fluorescent microplate reader – equipped with filter for Ex/Em = 480/511 nm

96 well plate: white plates for fluorometric assay

Vortex


GENERAL INFORMATION

1. LIMITATIONS Assay kit intended for research use only. Not for use in diagnostic

procedures.

Do not use kit or components if it has exceeded the expiration date on the kit labels.

Do not mix or substitute reagents or materials from other kit lots or vendors. Kits are QC tested as a set of components and performance cannot be guaranteed if utilized separately or substituted.


GENERAL INFORMATION

2. TECHNICAL HINTS This kit is sold based on number of tests. A ‘test’ simply

refers to a single assay well. The number of wells that contain sample, control or standard will vary by product. Review the protocol completely to confirm this kit meets your requirements. Please contact our Technical Support staff with any questions.

Keep enzymes and heat labile components and samples on ice during the assay.

Make sure all buffers and developing solutions are at room temperature before starting the experiment.

Avoid cross contamination of samples or reagents by changing tips between sample, standard and reagent additions.

Avoid foaming or bubbles when mixing or reconstituting components.

Samples generating values higher than the highest standard should be further diluted in the appropriate sample dilution buffers.

Ensure plates are properly sealed or covered during incubation steps.

Ensure complete removal of all solutions and buffers from tubes or plates during wash steps.

Make sure you have the appropriate type of plate for the detection method of choice.

Make sure the heat block/water bath and microplate reader are switched on before starting the experiment.


ASSAY PREPARATION

3. REAGENT PREPARATION Briefly centrifuge small vials at low speed prior to opening.

9.1 CETP Assay Buffer:Ready to use as supplied. Equilibrate to room temperature before use. Store at 4°C.

9.2 CETP Donor Molecule:Ready to use as supplied. Aliquot CETP donor molecule so that you have enough volume to perform the desired number of tests. Store at 4°C. Keep on ice while in use.

9.3 CETP Acceptor Molecule:Ready to use as supplied. Aliquot CETP acceptor molecule so that you have enough volume to perform the desired number of tests. Store at 4°C. Keep on ice while in use.

9.4 Positive Control (Rabbit serum):Ready to use as supplied. Aliquot positive control so that you have enough volume to perform the desired number of tests. Store at -80°C. Keep on ice while in use.

9.5 CETP Inhibitor (Torcetrapib):Ready to use as supplied. Aliquot inhibitor so that you have enough volume to perform the desired number of tests. Store at 4°C. Keep on ice while in use.


ASSAY PREPARATION

4. STANDARD PREPARATION Always prepare a fresh set of standards for every use.

To save time, the standard curve can be made during sample incubation.

10.1 Perform serial dilutions of the CETP Donor Molecule in 100% isopropanol as follows:

10.1.1 Dilute 10 µL of provided 8 nmol/mL Donor Molecule (Section 9.2) in 990 µL of 100% isopropanol = 80 pmol/mL CETP Donor Molecule standard.

10.1.2 Prepare a 20 pmol/mL Donor Molecule standard by adding 250 µL of standard from Section in 750 µL of 100% isopropanol = Standard #1.

10.1.3 Using Standard #1, prepare standard curve dilution as described in the table in a microplate or microcentrifuge tubes:

Standard#

Sample to dilute

Volume sample to dilute

(µL)

Volume of 100% IPP

(µL)

Final volume

standard in well (µl)

Final Con in well

1 Step 10.1.2 4 pmol2 Std #1 500 500 200 2 pmol3 Std #2 500 500 200 1 pmol4 Std #3 500 500 200 0.5 pmol5 Std #4 500 500 200 0.25 pmol6 - 0 500 200 0 pmol

Each dilution has enough amount of standard to set up duplicate readings (2 x 200 µL).Note: The Standard Curve wells should be read promptly after transferring the serially-diluted standards from microcentrifuge tubes to the 96-well plate, to minimize the evaporation of the isopropanol.

ASSAY PRE


ASSAY PREPARATION

5. SAMPLE PREPARATIONGeneral Sample information:

Collect plasma (recommended) or serum by standard methods.

We recommend that you use fresh samples. If you cannot perform the assay at the same time, store the samples immediately at -80°C. When you are ready to test your samples, thaw them on ice. Be aware however that this might affect the stability of your samples and the readings can be lower than expected.

11.1 Plasma or Serum:Collect plasma or serum by standard methods.Plasma and serum samples can be tested directly by adding sample to the microplate wells. Whenever possible, we recommend using plasma as preferred sample type.Dilute plasma or serum 1/5 – 1/10 in CETP Assay Buffer and use 2 – 10 µL to obtain signal within the range of the Standard Curve.Using higher than recommended amounts of plasma or serum will inhibit the signal (>2 µL undiluted).

NOTE: We suggest using different volumes of sample to ensure readings are within the Standard Curve range.

11.2 Positive Control (Rabbit Serum):Dilute rabbit Serum (Section 9.4) 1/10 in CETP Assay Buffer and use 10 µL of diluted positive control in the test.

11.3 Optional – Negative Control (Torcetrapib, 1mM):Dilute 4 µL Torcetrapib (Section 9.5) in 496 µL DMSO. Torcetrapib will inhibit both rabbit and human CETP.


ASSAY PROCEDURE and DETECTION

6. ASSAY PROCEDURE and DETECTION● Equilibrate all materials and prepared reagents to room

temperature prior to use.● It is recommended to assay all standards, controls and

samples in duplicate.12.1 Set up Reaction wells:- Standard wells = 200 µL standard dilutions.- Sample, background, positive and optional inhibitor wells as

follows:

12.2 Pre-incubate all wells (standard, samples and controls) at 37°C for 10 min protected from light to stabilize the signal.

12.3 Measure fluorescence on a microplate reader at Ex/Em = 488/523 nm in a kinetic mode, every 2 – 3 minutes, for 1 – 3 hours at 37°C.

NOTE: Sample incubation time can vary depending on CETP activity. We recommend measuring the fluorescence in kinetic mode and choosing two time points (T1 and T2) in the linear range to calculate the CETP activity of the samples. The Standard Curve can be read in the end point mode, although we suggest to read the standard no later than 1 hour.

ComponentSample

(µL)Background Control (µL)

Positive Control

(µL)Inhibitor ( µL)

CETP Donor Molecule 5 5 5 5CETP Acceptor Molecule 5 5 5 5

Sample (Section 11.1) 2 – 10 0 0 2 – 10Positive control (Section 11.2) 0 0 10 0

CETP Inhibitor (Section 11.3) 0 0 0 2

CETP Assay BufferAdjust volume to 200

Adjust volume to

200

Adjust volume to 200


ASSAY PRE


ASSAY PROCEDURE and DETECTION

High activity samples, such as rabbit serum, may have decreased activity rate after 1 hour. If you want to run the assay for a longer period, use less sample.


DATA ANALYSIS

7. CALCULATIONS Samples producing signals greater than that of the highest

standard should be further diluted in appropriate buffer and reanalyzed, then multiplying the concentration found by the appropriate dilution factor.

For statistical reasons, we recommend each sample should be assayed with a minimum of two replicates (duplicates).

Average the duplicate reading for each standard and sample. Subtract the mean absorbance value of the blank (Standard

#6) from all standard. This is the corrected absorbance. Plot the corrected absorbance values for each standard as a

function of the final concentration of Donor Molecule. Draw the best smooth curve through these points to construct

the standard curve. Most plate reader software or Excel can plot these values and curve fit. Calculate the trendline equation based on your standard curve data (use the equation that provides the most accurate fit).

Subtract reagent background control reading from sample reading and extrapolate from the standard curve plotted.

Activity of PLTP is calculated as:ΔRFU = (RFU2 – RFUBG2) – (RFU1 – RFUBG1)Where:RFU1 and RFU2 is the sample reading at time T1 and T2 respectivelyRFUBG1 and RFUBG2 is the reagent background control reading at time T1 and T2 respectively

Use the ΔRFU to obtain B pmol of cholesteryl ester transferred by CETP.

CETP activity (in pmol/µL/hr) in the test samples is calculated as:

𝐶𝐸𝑇𝑃 𝐴𝑐𝑡𝑖𝑣𝑖𝑡𝑦 = ( 𝐵Δ𝑇 𝑥 𝑉) ∗ 𝐷

= pmol/µL/hr


DATA ANALYSIS

Where: B = Amount of Cholesteryl Ester (Donor Molecule) from Standard Curve (pmol).ΔT = Reaction time (hour).V = sample volume added into the reaction well (µL).D = sample dilution factor.

Unit Definition:1 Unit CETP activity = amount of CETP that will transfer 1.0 µmol of Donor Molecule (Cholesteryl Ester) per hour at 37°C.


DATA ANALYSIS

8. TYPICAL DATATYPICAL STANDARD CURVE – Data provided for demonstration purposes only. A new standard curve must be generated for each assay performed.

Figure 1. Typical Donor Molecule standard calibration curve using fluorometric reading.


DATA ANALYSIS

Figure 2: Measurement of CETP activity of rabbit serum (1 µL), and Human plasma (4 µL).

Figure 3: Inhibition of CETP activity from rabbit serum by Torcetrapib. The assay was run for 1 hour and the IC50 was determined to be 3.56 nM.


DATA ANALYSIS

Figure 4: Inhibition of CETP activity from Human plasma using 80 nM Torcetrapib; assay was run for 2 hours.


RESOURCES

9. QUICK ASSAY PROCEDURENOTE: This procedure is provided as a quick reference for experienced users. Follow the detailed procedure when performing the assay for the first time.

Prepare standard, Donor Molecule, Acceptor Molecule, inhibitor and positive control (aliquot if necessary); get equipment ready.

Prepare standard curve using serial dilutions.

Prepare samples in duplicate (find optimal dilutions to fit standard curve readings).

Set up standard reaction wells – 200 µL

Set up rest of reaction wells as follows:

Pre-incubate 37°C for 30 min protected from light.

Measure fluorescence on a microplate reader at Ex/Em = 480/511 nm in a kinetic mode, every 2 – 3 minutes, for 1 – 3 hours at 37°C

Component Sample (µL)

Background Control (µL)

Positive Control

(µL)

Inhibitor ( µL)

CETP Donor Molecule 5 5 5 5CETP Acceptor Molecule

5 5 5 5

Sample, diluted 2 – 10 0 0 2 – 10Positive control, diluted

0 0 10 0

CETP Inhibitor 0 0 0 2CETP Assay Buffer Adjust

volume to 200

Adjust volume to

200




RESOURCES

10.TROUBLESHOOTING

Problem Cause Solution

Use of ice-cold buffer Buffers must be at room temperature

Plate read at incorrect wavelength

Check the wavelength and filter settings of instrument

Assay not

workingUse of a different 96-

well plate

Colorimetric: Clear platesFluorometric: black wells/clear

bottom plateSamples not

deproteinized (if indicated on protocol)

Use PCA precipitation protocol for deproteinization

Cells/tissue samples not homogenized

completely

Use Dounce homogenizer, increase number of strokes

Samples used after multiple free/ thaw

cycles

Aliquot and freeze samples if needed to use multiple times

Use of old or inappropriately stored

samples

Use fresh samples or store at - 80°C (after snap freeze in liquid

nitrogen) till use

Sample with erratic readings

Presence of interfering substance

in the sample

Check protocol for interfering substances; deproteinize samples

Improperly thawed components

Thaw all components completely and mix gently before use

Allowing reagents to sit for extended times

on ice

Always thaw and prepare fresh reaction mix before use

Lower/ Higher readings in samples and Standards Incorrect incubation

times or temperaturesVerify correct incubation times and temperatures in protocol


RESOURCES

Problem Cause SolutionPipetting errors in

standard or reaction mix

Avoid pipetting small volumes (< 5 µL) and prepare a master mix

whenever possibleAir bubbles formed in

wellPipette gently against the wall of

the tubes

Standard readings do not follow a linear pattern Standard stock is at

incorrect concentration

Always refer to dilutions on protocol

Measured at incorrect wavelength Check equipment and filter setting

Samples contain interfering

substances

Troubleshoot if it interferes with the kit

Unanticipated results

Sample readings above/ below the

linear range

Concentrate/ Dilute sample so it is within the linear range


RESOURCES

11.FAQ


RESOURCES

12.INTERFERENCES


RESOURCES

13.NOTES


RESOURCES


RESOURCES


RESOURCES

RESOURCES 27

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ab196995 II (Fluorometric CETP Activity Assay Kit...Ensure plates are properly sealed or covered during incubation steps. Ensure complete removal of all solutions and buffers from